Alzheimer's disease (AD) is a devastating neurodegenerative disease characterized by amyloid β (Aβ) plaque accumulation in brain regions involved in learning and memory. While these large insoluble plaques were once thought to cause AD, evidence now indicates that small diffusible oligomers of Aβ may be responsible. Amyloid-derived diffusible ligands (ADDLs) are a species of Aβ oligomers that can be generated in vitro with properties similar to endogenous Aβ oligomers (U.S. Pat. No. 6,218,506; Klein, et al. (2004) Neurobiol. Aging 25:569-580; Lambert, et al. (1998) Proc. Natl. Acad. Sci. USA 95:6448-6453). Aβ oligomers are present in the brain of AD patients, they bind neurons, and they induce deficits in neuronal morphology and memory. Studies with antibodies that bind Aβ oligomers have shown improvement in both neuronal morphology and memory.
Assays to measure Aβ monomers are known. For example, a sandwich ELISA composed of N-terminus (Aβ1) end-specific antibody (clone 82E1) and C-termini end-specific antibodies for Aβ1-40 (clone 1A10) and Aβ1-42 (clone 103) was developed to detect full-length Aβ1-40 and Aβ1-42 with a sensitivity in the sub-single digit fmol/ml (equivalent to single digit pg/ml) range with no cross-reactivity to APP (Horiskoshi, et al. (2004) Biochem. Biophys. Res. Commun. 319:733-737 and US Patent Publication No. 2011/0008339). Additional assays have used used the activity of β- and γ-secretase enzymes on the amyloid precursor protein (APP) to detect monomers; however, few assays have been reported that specfically and reliably detect Aβ oligomers in a human fluid sample, such as cerebrospinal fluid (CSF), in both normal control and in AD (Georganopoulou, et al. (2005) Proc. Nati. Acad. Sci. USA 102:2273-2276; Fukumoto, et al. (2010) FASEB J. 24:2716-2726; Gao, et al. (2010) PLoS One 5(12):e15725). Reported Aβ oligomer assays have employed a number of approaches, including ADDL-specific antibodies coupled with a bio-barcode PCR amplification platform (Georganopoulou, et al. (2005) supra), overlapping epitope ELISAs (Gandy, et al. (2010) Ann. Neurol. 68:220-230; Xia, et al. (2009) Arch. Neurol. 66:190-199), also paired first with size exclusion chromatography (Fukomoto, et al. (2010) supra), and amyloid-affinity matrices methods (Gao, et al. (2010) supra; Tanghe, et al. (2010) Int. J. Alz. Dis. 2010:417314), followed by oligomer dissociation and measurement with antibodies to Aβ monomers.
Aβ oligomers have also been detected from either CSF or brain using gel electrophoresis followed by western blot analysis (Klyubin, et al. (2008) J. Neurosci. 28:4231-4237; Hillen, et al. (2010) J. Neurosci. 30:10369-10379), or subsequent to size exclusion chromatography (Shankar, et al. (2011) Methods Mol. Biol. 670:33-44), relying on the molecular weight of oligomers that are maintained after the electrophoretic procedure. However, electrophoretic and blotting techniques do not provide the sensitivity required to see these species in normal control CSF (Klyubin, et al. (2008) supra), which exhibit a 1000-fold range of Aβ oligomer concentrations (Georganopoulou, et al. (2005) supra). Aβ oligomer species represent a wide range of molecular weights and, as such, assignment of a precise molarity is problematic. While a lower limit of detection at 100 aM has been shown (Georganopoulou, et al. (2005) supra), most reported methods (Georganopoulou, et al. (2005) supra; Gao, et al. (2010) supra; Fukumoto, et al. (2010) supra; Gandy, et al. (2010) supra) do not assess selectivity between signals from Aβ oligomers as compared to Aβ monomers, so the concentrations noted should be viewed with caution. One assay (Xia, et al. (2009) Arch. Neurol. 66:190-199), marketed by Immunobiological Laboratories, Inc. (Minneapolis, Minn.) claims 320-fold selectivity for Aβ1-16 dimers as compared to Aβ40 monomer, but lacks the selectivity needed to avoid cross-reactivity with Aβ monomer in the CSF. As Aβ oligomers in the CSF are hypothesized to be present at fM levels and CSF Aβ monomers are present between 1.5-2 nM, an assay that selectively measures Aβ oligomers in a CSF sample must have exceptional selectivity for Aβ oligomers over monomers.
Aβ oligomers have also been used as a target for therapeutic monoclonal antibodies to treat AD (see, for example, U.S. Pat. Nos. 7,811,563, 7,780,963, and 7,731,962). It is believed that these antibodies access the central nervous system (CNS) and clear the toxic ADDL species from the brain, through 1) catalytic turnover by Fc-mediated activation of microglia, 2) clearance of antibody/ADDL complexes into the cerebro-vasculature, or 3) enzymatic digestion of the ADDLs following antibody binding and improved access of degradative enzymes, such as neprilysin, insulin-degrading enzyme, plasmin, endothelin-converting enzymes (ECE-1 and -2), matrix metalloproteinases (MMP-2, -3 and -9), and angiotensin-converting enzyme (ACE). Thus, a goal of a selective Aβ oligomer assay is to measure the pharmacodynamic (PD) change in CNS Aβ oligomers following treatment with an anti-oligomer antibody or other treatment that alters Aβ monomer/oligomer formation or clearance. Additionally, an assay that would specifically enable the detection of Aβ oligomers bound to an anti-Aβ oligomer antibody, i.e., a target engagement (TE) assay, would be invaluable for the assessment of the therapeutic antibody following treatment.